Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma

BackgroundA major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients.Patients and methodsMatched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing.ResultsFor exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection.ConclusionsCombining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone.Clinical TrialsNCT01526928     

Super-ARMS法检测肺腺癌患者血浆循环肿瘤DNA表皮生长因子受体T790M基因突变的临床研究

  目的  探讨真实世界中Super-ARMS法检测肺腺癌患者外周血标本循环肿瘤脱氧核糖核酸（circulating tumor DNA,ctDNA）中表皮生长因子受体（epidermal growth factor receptor,EGFR）T790M基因突变的临床应用价值。  方法  收集2019年1月至2020年6月在首都医科大学附属北京胸科医院确诊的肺腺癌患者307例,突变扩增系统（amplification refractory mutation system, ARMS）检测组织中EGFR基因突变情况,Super-ARMS法检测血浆中EGFR的基因突变情况。通过生存分析比较不同标本检测EGFR T790M基因突变患者的无进展生存时间（progression-free survival,PFS）。  结果  153例患者疾病进展接受再活检。74例进行组织再活检,其中34例（45.9%）检测到EGFR T790M基因突变。141例患者进行液体再活检,其中51例（36.2%）EGFR T790M基因突变。Kaplan-Meier生存分析显示,组织和外周血EGFR T790M突变阳性患者接受第三代EGFR酪氨酸激酶抑制剂（EGFR-tyrosine kinase inhibitors,EGFR-TKIs）的中位PFS比较差异无统计学差异（16.3个月 vs. 11.4个月,x2=1.138,P>0.05）。组织和外周血EGFR T790M突变阴性患者未接受第三代EGFR-TKIs治疗的中位PFS比较差异无统计学意义（7.0个月 vs. 7.0个月,x2=0.470,P>0.05）。  结论  真实世界中Super-ARMS法检测外周血标本有望应用于检测EGFR T790M 基因突变情况,外周血标本可一定程度上补充组织标本检测EGFR T790M基因突变结果,预测患者对第三代EGFR-TKIs治疗的疗效。      

Detection of KRAS mutations in circulating tumour DNA from plasma and urine of patients with colorectal cancer

BackgroundCirculating tumour DNA (ctDNA) is very useful for purposes of cancer genetics; however, it has some limitations. Recently, ctDNA in body fluids, such as urine, sputum, and pleural effusion, has been investigated. The aim of this study was to evaluate the quantity of ctDNA derived from urine (trans-renal ctDNA) and the accuracy of KRAS mutation detection in relation to disease stage in colorectal cancer.MethodsUrine, plasma, and tissue samples were collected from consecutively resected colorectal cancer patients. DNA was extracted from each sample and the quantity was determined. From each DNA sample, KRAS mutations were detected using droplet digital PCR.Results200 patients participated and KRAS mutations were detected in 84 patients (42.0%) from tumour tissue. The concentration of trans-renal ctDNA (trtDNA) was significantly lower than that of plasma; however, there was no significant difference between the sensitivity using ctDNA and that using trtDNA (29.8% VS 33.3%, p = 0.62). Concordance between these two tests was only 17.5%. Combination analysis (ctDNA + trtDNA) improved the sensitivity to 53.6%, and sensitivity was significantly higher than that of corresponding single assays (p = 0.003). In early cancer stages, trtDNA had greater sensitivity for detecting KRAS mutations than ctDNA (37.7% vs. 21.3%, p = 0.047). Conversely, it was less useful for advanced cancer stages (21.7% vs. 52.2%, p = 0.07). Notably, KRAS mutations were detected using ctDNA or trtDNA in 12 of 116 (10.3%) patients who had no KRAS mutations in their tissue samples.Conclusions: trtDNA and ctDNA have equal potential and combination analysis significantly improved the sensitivity.     

EGFR酪氨酸激酶区域突变对NSCLC放疗敏感性影响的初步研究

目的:探讨非小细胞肺癌(NSCLC)组织中表皮生长因子受体(EGFR)酪氨酸激酶区域突变能否影响放射治疗疗效。方法:选取野生型EGFR和突变型EGFR细胞株A549和H1975,分别进行不同剂量的X射线照射后,用克隆细胞计数的方法计算细胞生存分数(SF),并采用SPSS 13.0软件按线性二次模型拟合细胞存活曲线,计算α/β值。免疫荧光技术观察磷酸化H2AX(γH2AX)焦点数的变化,推测DNA双链断裂后修复动力学。结果:H1975及A549细胞SF2值分别为0.62和0.89,α/β分别为4.00和0.19。H1975细胞SF2较A549小,差异有统计学意义,P<0.05;但α/β值较大,差异有统计学意义,P<0.05。2种细胞株在照射4Gy后15min开始出现γH2AX焦点数增多,但是H1975细胞株较A549多,于3h时保持约70%,24h时仍有约25%,而A549细胞株在照射后3h消失接近90%。结论:NSCLCEGFR酪氨酸激酶区域突变能增加放射敏感性,可成为预测放射治疗疗效的一个指标。     

The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies

To understand the role of human epidermal growth factor receptor (hEGFR) kinase domain mutations in lung tumorigenesis and response to EGFR-targeted therapies, we generated bitransgenic mice with inducible expression in type II pneumocytes of two common hEGFR mutants seen in human lung cancer. Both bitransgenic lines developed lung adenocarcinoma after sustained hEGFR mutant expression, confirming their oncogenic potential. Maintenance of these lung tumors was dependent on continued expression of the EGFR mutants. Treatment with small molecule inhibitors (erlotinib or HKI-272) as well as prolonged treatment with a humanized anti-hEGFR antibody (cetuximab) led to dramatic tumor regression. These data suggest that persistent EGFR signaling is required for tumor maintenance in human lung adenocarcinomas expressing EGFR mutants.     

Heterogeneity in the clinical phenotype of TP53 mutations in breast cancer patients.

TP53 mutation is a strong independent marker for survival in breast cancer with some heterogeneity in the clinical phenotype of various types of mutations. Based on 315 patients with breast carcinoma, we suggest a new model for the differentiation of TP53 mutations. Although TP53 mutation in general was associated with aggressive tumor/patient characteristics, missense mutations outside any conserved or structural domain did not affect the clinical outcome (risk of disseminated disease and death). In contrast, patients with missense mutations affecting amino acids directly involved in DNA or zinc binding displayed a very aggressive clinical phenotype. Null mutations (including missense mutations disrupting the tetramerization domain) and the remaining missense mutations displayed an intermediate aggressive clinical phenotype. When patients with primary early breast cancer were divided into three groups (wild-type together with missense mutations outside structural/conserved domains, null mutations and missense mutations with intermediate clinical phenotype, and very aggressive missense mutations), disease-specific survival rates were 89%, 58%, and 35% (5-year actuarial values, P < 0.0001), respectively. In a Cox proportional hazards analysis, separation of TP53 mutations according to these criteria eliminated the prognostic importance of all investigated classical factors except nodal status.     

Silencing Ku80 using small interfering RNA enhanced radiation sensitivity in vitro and in vivo

Ku80 is an important component of DNA double-strand break repair, and Ku80 deficiency leads to extreme sensitivity to ionizing radiation. We studied whether radiation therapy combined with Ku80 silencing by small interfering RNA enhances radiation sensitivity in vitro and in vivo. Seven human cancer cell lines were transfected with Ku80 siRNA included in hemagglutinating virus of Japan envelope vector. H1299 cells were implanted into male BALB/C nu/nu nude mice treated with Ku80 siRNA and irradiation. The survival rate of cell lines transfected with Ku80 siRNA decreased by 10% to 26% with 2-Gy irradiation compared with untransfected cell lines. The gamma-H2AX phosphorylation-positive rates of Ku80 siRNA combined treatment 0.5 h after irradiation in A549 cells and 6 h in H1299 cells were significantly higher (77.6%, p=0.033 and 76.7%, p=0.026, respectively), compared with the groups not treated with siRNA. H1299 xenograft tumors treated with combined therapy decreased in volume and re-grew slowly compared with radiation alone. Our results indicate that combined therapy consisting of Ku80 siRNA and irradiation contributes to inhibition of tumor growth and may be a novel strategy for cancer treatment.     

Dynamics of circulating tumor DNA represented by the activating and resistant mutations in epidermal growth factor receptor tyrosine kinase inhibitor treatment

Circulating tumor DNA (ctDNA) is an emerging field of cancer research. For lung cancer, non‐invasive genotyping of EGFR is the foremost application. The activating mutations represent the ctDNA from all cancer cells, and the T790M‐resistant mutation represents that from resistant cells. We examined the ctDNA dynamics of EGFR mutations by using deep sequencing with a massively parallel DNA sequencer. We obtained 190 plasma samples from 57 patients at various times during the treatment course and classified them according to treatment status. The mutation detection rate of exon 19 deletion/L858R in plasma was high at the initiation of treatment with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI; P = 0.001), suppressed during EGFR‐TKI treatment before disease progression, and elevated after the onset of disease progression (P = 0.023). The mutation detection rate of T790M was low until the onset of disease progression and elevated thereafter (P = 0.01). Samples across the development of disease progression were obtained from 10 patients and showed a correlation between increased ctDNA level and disease progression. Decreased ctDNA level in response to the initiation of EGFR‐TKI was observed in 4 of 6 eligible patients. In two patients, the ctDNA dynamics suggested the presence of cancer cell populations only with the T790M mutation. In another patient, the T790M ctDNA represented cell subpopulations that respond to cytotoxic agents differently from the major population. Considering the high incidence, ctDNA could be a clinical parameter to complement information from image analyses.     

Heterogeneity and phenotypic instability of chemotherapeutic and immunologic sensitivity in murine and human melanoma cell clones.

The aim of the present study was to examine the phenotypic heterogeneity of murine and human melanoma cell lines with particular reference to anticancer drug sensitivity, growth pattern and susceptibility to lysis by lymphokine (rIL2) activated killer (LAK cells). Clones selected for a different drug sensitivity were tested to evaluate the stability of such properties after different in vitro passages. A possible relationship between drug sensitivity and LAK susceptibility was also analyzed. The results indicated a high heterogeneity in murine and in human melanoma clones for all the parameters. However, drug sensitivity, which was stable although for only a few passages in an untreated human melanoma, was highly unstable in murine naturally or drug-induced resistant cells. Finally, whereas human drug-resistant clones were sensitive to lysis by LAK cells and an inverse correlation was found with the level of drug resistance, murine clones appeared to be LAK sensitive, and no correlation was found between the level of drug resistance and LAK sensitivity. Our data indicate a different stability in drug response of human and murine cells and a different behaviour of human and murine drug-resistant cells in response to LAK lysis.     

Novel Abl kinase inhibitors in chronic myeloid leukemia in blastic phase and Philadelphia chromosome-positive acute lymphoblastic leukemia

Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia chromosome, which is associated with a balanced translocation involving chromosomes 9 and 22 to produce a fusion gene (bcr-abl) that gives rise to a constitutively activated Abl tyrosine kinase. This kinase led to the discovery of several small-molecule inhibitors, imatinib being the first and most successful of these. Resistance to imatinib results in some patients from Abl kinase point mutations. Overcoming imatinib resistance represents one of the biggest challenges facing clinicians in the modern management of CML. In this review, we discuss the current understanding of CML pathophysiology and mechanisms of imatinib resistance and how advancing this knowledge has led to the design of novel therapies in the area of blastic phase CML and Philadelphia chromosome-positive acute lymphoblastic leukemia with previous imatinib failure.     

定量PCR检测肝细胞癌患者血浆循环DNA及其临床意义

背景与目的:血循环DNA是一种新的肿瘤诊断及预后判断的标志物.本研究运用定量PCR技术检测肝细胞癌(hepatocellular carcinoma,HCC)患者血浆循环DNA含量并探讨其诊断价值.方法:收集72例HCC患者术前血浆样本,37例肝良性病变(肝硬化以及慢性肝炎)和41例健康志愿者的血浆样本,纯化血浆循环DNA,采用实时定量PCR技术对血浆DNA水平进行检测.应用接受者操作特性(receiver-operating characteristics,ROC)曲线分析血浆循环DNA在HCC诊断中的价值.结果:HCC中位血浆循环DNA浓度(173 ng/mL)显著高于健康对照(9 ng/mL)和肝良性病变组(46 ng/mL)(P<0.001);其ROC曲线下面积(area under the ROC curve,AUC)分别为0.949和0.874.而HCC血浆DNA浓度也显著高于肝硬化及慢性肝炎患者(P=0.001),AUC为0.703.以18.2 ng/mL作为诊断HCC的临界值,其诊断特异度为90.2%,敏感度达90.3%;与血清AFP联合检测可提高HCC诊断效率,AUC上升至0.974,其诊断特异度和敏感度分别为95.1%和94.4%.伴肝内播散或脉管癌栓HCC患者的血浆DNA浓度(261 ng/mL)明显高于不伴肝内播散灶或脉管痛栓患者(142 ng/mL,P=0.035).结论:定量PCR技术可精确定量血浆循环DNA浓度;血浆DNA分析对于HCC诊断,预测肿瘤转移潜能具有重要价值.     

Highly sensitive fusion detection using plasma cell-free RNA in non-small-cell lung cancers

ALK, ROS1, and RET kinase fusions are important predictive biomarkers of tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). Analysis of cell-free DNA (cfDNA) provides a noninvasive method to identify gene changes in tumor cells. The present study sought to use cfRNA and cfDNA for identifying fusion genes. A reliable protocol was established to detect fusion genes using cfRNA and assessed the analytical validity and clinical usefulness in 30 samples from 20 cases of fusion-positive NSCLC. The results of cfRNA-based assays were compared with tissue biopsy and cfDNA-based liquid biopsy (Guardant360 plasma next-generation sequencing [NGS] assay). The overall sensitivity of the cfRNA-based assay was 26.7% (8/30) and that of cfDNA-based assay was 16.7% (3/18). When analysis was limited to the samples collected at chemo-naïve or progressive disease status and available for both assays, the sensitivity of the cfRNA-based assay was 77.8% (7/9) and that of cfDNA-based assay was 33.3% (3/9). Fusion gene identification in cfRNA was correlated with treatment response. These results suggest that the proposed cfRNA assay is a useful diagnostic test for patients with insufficient tissues to facilitate effective administration of first-line treatment and is a useful tool to monitor the progression of NSCLC for consideration of second-line treatments.     

Focal adhesion kinase targeting using in vivo short interfering RNA delivery in neutral liposomes for ovarian carcinoma therapy.

PURPOSE: Focal adhesion kinase (FAK) plays a critical role in ovarian cancer cell survival and in various steps in the metastatic cascade. Based on encouraging in vitro results with FAK silencing, we examined the in vivo therapeutic potential of this approach using short interfering RNA (siRNA) in the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). EXPERIMENTAL DESIGN: Therapy experiments of FAK siRNA with or without docetaxel were done using human ovarian cancer cell lines SKOV3ip1, HeyA8, and HeyA8MDR in nude mice. Additional experiments with a cisplatin-resistant cell line (A2780-CP20) were also done. Assessments of angiogenesis (CD31), cell proliferation (proliferating cell nuclear antigen), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) were done using immunohistochemical analysis. RESULTS: A single dose of FAK siRNA-DOPC was highly effective in reducing in vivo FAK expression for up to 4 days as assayed by Western blot and immunohistochemical analysis. Therapy experiments were started 1 week after injection of the ovarian cancer cells. Treatment with FAK siRNA-DOPC (150 mug/kg twice weekly) reduced mean tumor weight by 44% to 72% in the three cell lines compared with the control group (Ps < 0.05 for HeyA8, A2780-CP20, and SKOV3ip1). When FAK siRNA-DOPC was combined with docetaxel, there was even greater reduction in mean tumor weight in all models (all Ps < 0.05). Similar results were observed in combination with cisplatin. Treatment with FAK siRNA-DOPC plus docetaxel resulted in decreased microvessel density, decreased expression of vascular endothelial growth factor and matrix metalloproteinase-9, and increased apoptosis of tumor-associated endothelial cells and tumor cells. CONCLUSIONS: Taken together, these findings suggest that FAK siRNA-DOPC plus docetaxel or platinum might be a novel therapeutic approach against ovarian cancer.     

Reciprocal regulation of Abl kinase by Crk Y251 and Abi1 controls invasive phenotypes in glioblastoma

Crk is the prototypical member of a class of Src homology 2 (SH2) and Src homology 3 (SH3) domain-containing adaptor proteins that positively regulate cell motility via the activation of Rac1 and, in certain tumor types such as GBM, can promote cell invasion and metastasis by mechanisms that are not well understood. Here we demonstrate that Crk, via its phosphorylation at Tyr251, promotes invasive behavior of tumor cells, is a prominent feature in GBM, and correlating with aggressive glioma grade IV staging and overall poor survival outcomes. At the molecular level, Tyr251 phosphorylation of Crk is negatively regulated by Abi1, which competes for Crk binding to Abl and attenuates Abl transactivation. Together, these results show that Crk and Abi1 have reciprocal biological effects and act as a molecular rheostat to control Abl activation and cell invasion. Finally, these data suggest that Crk Tyr251 phosphorylation regulate invasive cell phenotypes and may serve as a biomarker for aggressive GBM.